Ornithobacterium rhinotracheale vaccine and method of immunization

ABSTRACT

The present invention relates to a novel bacterial respiratory poultry disease and the identification of the causative agent Ornithobacterium rhinotracheale. A vaccine derived from this agent was effective in preventing the disease in chickens challenged with the virulent field strains.

The present invention is concerned with a novel type of gram-negativeaerobic rod-shaped bacterium, a vaccine derived thereof and with the useof a novel type of Gram-negative aerobic rod-like bacterium. Thisbacterium has been classified as a new genus and species, and accordedthe name Ornithobacterium rhinotracheale.

In the last decades, in many countries a strong rise in both the numberof chicken and poultry farms, and in addition, an increasing number ofanimals per farm has been seen. This situation has a seriousconsequence: it has caused an increasing need for new and bettervaccines and vaccination programmes in these countries. Nowadays, mostanimals are immunized against a number of diseases of viral, bacterialand parasitic origin. Examples of viral diseases in poultry areNewcastle Disease, Infectious Bronchitis, Turkey Rhinotracheitis,Herpesvirus of Turkeys, Fowlpox, Infectious Bursal Disease, etc.Examples of bacterial diseases are Coryza, Salmonella infections,Pasteurella multocida infections and E. coli infections.

A new bacterial respiratory disease has surprisingly been observed inchickens and turkeys. The disease was seen in chickens that had beenvaccinated against the bacterium Haemophilus paragallinarum; thecausative agent of a disease called Coryza. Coryza is, as far as known,the only respiratory disease in chicken, caused by bacteria belonging tothe families of Pasteurellaceae and Neisseriaceae. The symptoms of thisnew disease differ from the specific symptoms of Coryza. Coryza ismainly an infection of the upper respiratory tract. Infected animalsshow a serous to mucoid nasal discharge, facial edema andconjunctivitis. They do however not show the clinical signs belonging todiseases of the lower respiratory tract as e.g. airsacculitis orcoughing, pneumonic lungs or pleuritis. Given the fact that the newlydiscovered disease clearly shows the clinical signs of a lowerrespiratory tract infection as described below, H. paragallinarum couldbe ruled out as the causative agent.

The newly discovered disease is characterised by the following clinicalsigns in chickens: The first indication of this new disease is mildsnicking. Two or tree days later a small number of broilers usuallydevelop a mild nasal discharge and/or mild facial edema, which disappearafter 2-4 days. Snicking continues until the birds are processed. Within1-3 days from the beginning of respiratory symptoms, evidence of areduction in feed intake can be detected. This is associated with someincreased mortality mainly from broilers succumbing with pneumonic lungsand pleuritis, often with thickened thoracic airsacs. From theselesions, E. coli is the dominant isolate. Subsequent losses are mainlyassociated with extensive airsacculitis. Examination of live broilers atthe start of the syndrome usually reveals no specific pathology. Fromsinuses of affected broilers a pasteurella-like organism canoccasionally be isolated. After a couple of days, 30-60 % of thebroilers suffer from extensive involvement of especially the abdominaland thoracic airsacs. Especially noticeable is the severe thickening ofthe airsac membranes. These airsacs often contain a copious amount of acreamy white-yellowish exudate. A somewhat velvety appearance of theairsac is also common. A whitish-creamy foamy exudate is often evidenton the mesentery as well. Histopathology reveals a prominent exudativeinflammatory process with a fibrinous exudate on the surface and withinthe membrane, with oedema as well. Accumulation of plasma cells andheterophils are noticeable with some multi-nuclear giant cells andgranulomatous infiltrations. No specific micro-organisms are visible insections with Ziehl-Nielsen and PAS staining. In live birds nopericarditis, perihepatitis or splenitis is usually seen. From affectedairsacs Pasteurella/Neisseria-like organisms were isolated. Theseisolates did not seem to be classic species in the sense that they do inspite of their relatedness to Pasteurella and Neisseria, not belong tothese species and some variation in their biochemical abilities has beennoticed.

In turkey flocks in several parts of the world, a comparable infectionof the upper respiratory tract was found. At first appearance, a lowmortality wax found, although at this moment mortality in flockssuffering from the disease can be as high as 5%. The first clinicalsigns are comparable to infection in chicken: sneezing and nasaldischarge. In some animals clinical signs of acute infection were seen.Examination of sacrificed animals showed edema of the lungs,fibrinopurulent pneumonia and often serofibrinous pericarditis andserofibrinous infection of the airsacs.

Bacteria were isolated from infected airsacs and purified. Afterpurification, isolates were grown on rich agar dishes in order to obtainlarge quantities of pure pathogen. In order to check for the validity ofthe Koch postulates, a group of S(pecific) P(athogen) F(ree) animals wasinfected with a mixture of isolates. After infection, they all showedclinical signs that were indistinguishable from those, seen in fieldinfections. From the airsacs of these infected animals, bacteria wereisolated that were serologically indistinguishable from the challengestrains.

Four similar highly homologous strains were isolated from the airsacs ofsick chickens. One very similar strain was isolated from the airsacs ofturkeys. The strains were identified as being Gram-negative aerobicrods. The various isolates show minor differences in their respectivefermentation-patterns. All strains however clearly belong to the sameserotype, i.e. serum raised against each of the five strains(cross-)reacted with each of the strains. One of the chicken-strains isdeposited at the Centraalbureau voor Schimmelcultures (CBS),Oosterstraat 1, PO.box 273, 3740 AG Baarn, The Netherlands, underaccession-number 400.92.

The invention provides a novel type of gram-negative aerobic rod-shapedbacterium, said novel type of bacterium being characterised by thebacterium deposited at the Centraalbureau voor Schimmelcultures underdeposit number 400.92 the date of deposit being Sep. 8, 1992.

With the wording "bacteria of a novel type" is meant gram-negativeaerobic rod-shaped bacteria that are serologically related to thedeposited strain. Serologically related bacteria are bacteria thatdisplay cross-reactivity with sera raised against the deposited strain.

In particular, gram-negative aerobic rod-shaped bacteria are envisagedthat give a higher titer, in serological tests, with antiserum againstthe deposited strain than with antisera against known gram-negativeaerobic rod-shaped bacteria.

More in particular, the present invention is directed to gram-negativeaerobic rod-shaped bacteria that positively react in an-Agar GelPrecipitation test with antiserum derived against the deposited strain.

The deposited bacterium was typed according to standard determinationmethods, using Bergey's Manual of Systematic Bacteriology Volume 1(1984, Williams and Wilkins, 428 East Preston Street, Baltimore U.S.A.,1984) and A.P.I SYSTEM, La Balme-les-Grottes 38390 Montalieu-Vercie,France, system numbers API 20E, API 20NE, API 50CHE, API ZYM, API OF.Results are shown in table 1.

                  TABLE 1                                                         ______________________________________                                        differentiation tests.                                                        ______________________________________                                        nitrate reduction   -                                                         V-factor requirement                                                                              -                                                         catalase            -                                                         cytochrome-oxidase  +                                                         growth on McConkey-agar                                                                           -                                                         Voges Proskauertest (37° C.)                                                               + (weakly)                                                Urease              +                                                         lysine decarboxylase                                                                              -                                                         ornithine decarboxylase                                                                           -                                                         O.N.P.G. or P.N.P.G. (β-gal)                                                                 +                                                         strictly aerobic    -                                                         arginine dehydrolase                                                                              + (temp.-dependent)                                       indole              -                                                         fermentation of:                                                              fructose            +                                                         lactose             +                                                         galactose           +                                                         ______________________________________                                    

The combination of characteristic properties as given in table 1 makesthe novel type of bacteria unique compared to other known bacterialpoultry pathogens. (Diseases of Poultry 8, Iowa State University Press1984). Incidentally, a strain according to the invention may reactnegatively in a test of table 1, where the deposited strain reactspositively, or vice versa. This is especially the case when the reactionis varying between weakly positive and negative. This may be due tosmall differences between the tested strains; slight differences areinherent to biological systems. It may also be due to small differencesin the reaction conditions in various test-labs.

Several chicken strains with the characteristics of the depositedbacterium have been isolated from chickens suffering from the diseasedescribed above, and antisera induced after challenge with livepathogens in Specific Pathogen Free chickens have been checked for(cross-)reaction with the isolated strains. As is shown in table 2,antiserum raised against each strain as determined by an ELISA-methodusing boiled capsular antigen according to the method of Heddleston, K.L. et al. (Avian Diseases 16:925 (1972)) gives a positive reactioni.e. >6 with all other strains.

                  TABLE 2                                                         ______________________________________                                        (cross-)reactivity of the deposited strain and                                three homologous strains, determined by ELISA.                                titer against strain                                                          Challenge                                                                              3037/91   3263/91 3290/91/(A)                                                                            3290/91/(K)                               ______________________________________                                        3037/91   7        7        8        7                                         3263/91 13       12       >13      13                                        3290/91(A)                                                                              9        9       11       10                                        3290/91(K)                                                                             13       12       12       12                                        ______________________________________                                          The underlined strain is of the bacterium, deposited under nr. CBS           400.92-                                                                  

Pooled sera of groups of broilers, vaccinated with one of the strainsgiven in the table, (vaccines prepared as in Example I), were tested inthe ELISA test described above for (cross-)reactivity. Titers wereraised after repeated vaccination in the presence of adjuvant. StrainGGD 1261 is a strain, recently isolated from turkeys by Dr H. M. Hafez,State Veterinary Laboratory of Stuttgart Germany). As is clearly shownin table 3, all strains are (cross-)reactive, although strainsoriginating from chickens react better with antisera against chickenstrains, and the turkey-strain react better with antisera against theturkey strain.

                                      TABLE 3                                     __________________________________________________________________________    serological responses, after vaccination, of the combined sera                of the groups against boiled capsular extracts. Serum taken 3 weeks           after 2.sup.nd vaccination.                                                                boiled capsular extracts prepared from:                          Serum nr.                                                                           vacc. with                                                                           3037/91                                                                            3263/91                                                                            3290/91(A)                                                                           3290/91(K)                                                                           GGD-1261                                 __________________________________________________________________________    612   CONTROL                                                                               <6   7    <6     <6    <6                                       613   3037/91                                                                              >19  >19  >19    >19    11                                       614   3263/91                                                                              >19  >19  >19    >19    17                                       615   3290/91(A)                                                                            19   19   19    >19    13                                       616   3290/91(K)                                                                            19   19   19    >19    14                                       619   GGD-1261                                                                              10   11   11     12    >19                                      __________________________________________________________________________     The underlined strain is of the bacterium deposited under nr. CBS 40092  

It is obvious, that any strain isolatable from airsacs of animalssuffering from the described illness and serologically related to thedeposit strain also falls within the scope of the present invention.Thus, the novel type of bacterium comprises bacteria which arecross-reactive with the deposited bacterial strain, i.e. serum raisedagainst a novel type bacterium binds to the deposited bacterium and viceversa. In order to discriminate between the novel type of bacterium ofthe present invention and other gram-negative aerobic rod-shapedbacteria, two serological tests were done:

a) the strain of the present invention was tested in an Agar GelPrecipitation test according to Heddlestone (Heddleston, K. L. et al.(Avian Diseases 16:925 (1972)) against strain 3037/91, strain3290/91(A), strain 3290/91(K), all isolated from chickens, and strainGGD1261, isolated from turkey. In all cases, cross-reaction was found.The strain of the present invention was also tested with Haemophilusparagallinarum strains H18, Spross, 0083, against Kingella kingae, andKingella denitrificans, against Suttonella indologenes, againstPasteurella gallinarum, against the known 16 serotypes of Pasteurellamultocida and against 10 serotypes of Pasteurella anatipestifer. Nocross-reactivity was found.

b) The strains mentioned in Table 3 were tested in an ELISA assayagainst three different serotypes of Haemophilus paragallinarum, againsttwo Kingella strains, against Suttonella indologenes and againstPasteurella gallinarum. The results, given in table 4 show that,although the cross-reactivity between related strains is (very) high,there is no cross-reactivity between any of the strains from table 3 andthe known strains listed in table 4.

                                      TABLE 4a                                    __________________________________________________________________________    SEROLOGICAL RESPONSES OF THE COMBINED SERA OF THE GROUPS                      AGAINST BOILED CAPSULAR EXTRACTS SERUM TAKEN 3 WEEKS AFTER                    2nd VACCINATION.                                                              Sera with a titer of 10 or >10 are considered to belong to to the same        serotype.                                                                     SERUM                                                                              VACC.  TITRE (IN 2 LOG) AGAINST B.C.A. OF STRAIN;                        NR   WITH   3037/91                                                                            3263/91                                                                            3290/91(A)                                                                           3290/91(K)                                                                           GGD-1261                                  __________________________________________________________________________    612  control                                                                              <6    7   <6     <6     <6                                        613  3037/91                                                                              >19  >19  >19    >19    11                                        614  3263/91                                                                              >19  >19  >19    >19    17                                        615  3290/91(A)                                                                           19   19   19     >19    13                                        618  3290/91(K)                                                                           19   19   19     >19    14                                        619  GGD-1261                                                                             10   11   11     12     >19                                       620  Hpg-H18                                                                               8    9    8      8      8                                        621  Hpg-Spross                                                                            7    8    8      8      8                                        622  Hpg-0083                                                                              7    8    8      8      7                                        628  K. kingae                                                                             7    8    8      7      7                                        629  K. denitr.                                                                            7    9    8      9      7                                        630  S. indolog.                                                                           6    7    8      7      6                                        631  P. gallin.                                                                            6    8    7      7      6                                        __________________________________________________________________________

                                      TABLE 4b                                    __________________________________________________________________________    SEROLOGICAL RESPONSES OF THE COMBINED SERA OF THE                             GROUPS AGAINST BOILED CAPSULAR EXTRACTS SERUM TAKEN                           3 WEEKS AFTER 2nd VACCINATION.                                                Sera with a titer of 10 or >10 are considered to belong to to the same        serotype.                                                                                 TITRE (IN 2 LOG) AGAINST B.C.A. OF STRAIN;                        SERUM                                                                              VACC.  Hpg-                                                                             Hpg- Hpg-                                                                             K.  K.  S.    P                                        NR   WITH   H18                                                                              SPROSS                                                                             0083                                                                             kingae                                                                            denitr.                                                                           indolog.                                                                            gallin.                                  __________________________________________________________________________    612  control                                                                              <6 <6   <8 <6  8   6    <6                                        613  3037/91                                                                              7  6    6  <9  <9  <9   6                                         614  3263/91                                                                              <6 6    <6 <9  <9  <9   6                                         615  3290/91(A)                                                                           7  8    7  <9  <9  <9   7                                         618  3290/91(K)                                                                           6  8    6  <9  <9  <9   7                                         619  GGD-1261                                                                             6  7    6  <6  7   7    7                                         620  Hpg-H18                                                                              >13                                                                              13   10 8   12  11   9                                         621  Hpg-Spross                                                                           11 >13  >13                                                                              8   12  11   9                                         622  Hpg-0083                                                                             11 13   >13                                                                              9   11  11   8                                         628  K. kingae                                                                            8  9    7  >15 15  7    7                                         629  K. denitr.                                                                           9  9    7  12  >15 7    7                                         630  S. indolog.                                                                          6  6    <6 6   6   >15  6                                         631  P. gallin.                                                                           8  9    8  8   8   8    >13                                       __________________________________________________________________________

Preferably, the invention provides bacteria of a novel type as definedabove, further characterised in that they display the biochemicalproperties given in table 1.

In particular, the present invention provides a specific strain of thenovel bacterium, i.e. the strain deposited at the Centraalbureau voorSchimmelcultures (CBS), Oosterstraat 1, PO.box 273, 3740 AG Baarn, TheNetherlands, under accession-number 400.92.

The invention also relates to a microbiological culture comprising abacterium of the novel type as described above. The culture may be madeby growing said bacteria at a temperature of between 30° and 41° C. Thebacteria may be grown under normal atmospheric oxygen pressure whereasthe percentage of CO₂ in the environment is preferentially kept between0% and 10%. The bacteria can be grown in a variety of different generalpurpose bacterial growth promoting media e.g. Luria Broth or Brain HeartInfusion broth. The bacteria may also be grown on eggs, e.g. embryonatedchicken or turkey eggs. These eggs may be incubated preferentiallybetween 35° and 40° C.

The invention further provides a vaccine derived from the newlyidentified bacteria defined above.

Preferably, the invention provides a vaccine comprising bacteria derivedfrom the strain deposited with the CBS under nr. 400.92.

The vaccine according to the invention may comprise the bacteria in liveor attenuated live or inactivated form. Furthermore, fractions of wholecells may also be used as the relevant immunogen in the vaccineaccording to the invention.

In a preferred embodiment, said vaccine comprises inactivated bacteria.Various physical and chemical methods of inactivation are known in theart. Examples of physical inactivation are UV-radiation, X-rayradiation, gamma-radiation and heating. Examples of inactivatingchemicals are β-propiolactone, glutaraldehyde, ethyleneimine andformaldehyde.

Preferably the strain is inactivated with formaldehyde. It is obviousthat other ways of inactivating the bacteria are also embodied in thepresent invention.

The vaccine according to the invention in a preferred presentation alsocomprises an adjuvant. Adjuvants in general comprise substances thatboost the immune response of the injected animal. A number of differentadjuvants are known in the art. Examples of adjuvants are FreundsComplete and Incomplete adjuvant, vitamin E, non-ionic block polymers,muramyldipeptides, Quill A, mineral oil, vegetable oil, and Carbopol (ahomopolymer). In addition, the vaccine may comprise one or more suitableemulsifiers, e.g. Span or Tween.

In a preferred embodiment, the bacterin comprises a water-in-oilemulsion adjuvant. It goes without saying, that other ways of adjuvatingthe bacteria are also embodied in the present invention.

The vaccine in the present invention contains at least one antigen of abacterium of the novel type characterised by the bacterium depositedunder CBS 400.92. This includes whole cells, bacterial extracts, OuterMembrane Fractions, bacterial exo- and/or endotoxins, and purifiedproteins. It is obvious that antigenic polypeptides or fragments thereofmay for example be obtained from purified bacterial proteins or byexpression of the corresponding genetic material in some pro- oreu-karyotic expression-system or by organo-chemical synthesis.

The vaccine in the present invention may in addition to antigens of thenovel bacteria also contain antigenic material of at least one of theviruses and/or microorganisms of the group of poultry-pathogens,preferably in the form of live or inactivated viruses or microorganisms.

In a more preferred embodiment, said vaccine also comprises antigenicmaterial of the viruses or bacteria selected from, but not restrictedto, the group consisting of Infectious Bronchitisvirus, NewcastleDiseasevirus, Infectious Bursal Diseasevirus (Gumboro), Chicken Anaemiaagent, Avian Reovirus, Mycoplasma gallisepticum, TurkeyRhinotracheitisvirus, Haemophilus paragallinarum (Coryza), ChickenPoxvirus, Avian Encephalomyelitisvirus, Fowl Cholera and E. coli.

The present invention also relates to the use of bacteria of the noveltype for the preparation of a vaccine for the prevention of respiratorydiseases.

EXAMPLE I Growth of the novel bacteria, preparation of the vaccine andvaccination of broilers

Cells of the highly identical isolates strain 3037/91, 3263/91(deposited strain CBS 400.92), 3290/91(A) and 3290/91(K) were grown onsheepblood-agar for 48 hours at 37° C. with the use of a Gas-pac systemin order to obtain a 5-10% CO₂ environment. Cells were washed off and aC(olony) F(orming) U(nits) count was performed. Cells were killed byadding formaldehyde to a final concentration of 0.185%. After asterility-check, cells were diluted to 5*10⁸ C.F.U./cell-type in 1 ml ofthe final vaccine.

The vaccine was prepared by mixing the four strains and oil-adjuvant (awater-in-oil emulsion on the basis of a mineral oil with a ratio of 55%oil/45% water) to a final concentration of 5*10⁸ cells/strain/ml.

Vaccination was done in broilers at ten days of age and was performed byinjection of 0.5 ml of the vaccine subcutaneously halfway the neck.

EXAMPLE II Preparation of challenge strains and challenge of vaccinatedand control groups

Preparation 1): bacterial strains 3037/91, 3263/91, 3290/91(A) and3290/91(K) were grown in Brain Heart Infusion broth, for 20 hrs at 37°C. For challenge, preparations were made that contain the followingnumber of cells in the final challenge-volume:

    3.4*10.sup.8 c.f.u. of strain 3037/91

    2.2*10.sup.8 c.f.u. of strain 3263/91

    3.4*10.sup.8 c.f.u. of strain 3290/91(A)

    7.0*10.sup.7 c.f.u. of strain 3290/91(K)

Preparation 2): embryonated eggs were inoculated with bacterial strains3037/91, 3263/91, 3290/91(A) and 3290/91(K). The eggs were incubated at37° C. and egg-yolk was harvested after 2 days. For challenge,preparations were made that contain the following number of cells in thefinal challenge-volume:

    3.6*10.sup.6 c.f.u. of strain 3037/91

    6.6*10.sup.7 c.f.u. of strain 3263/91

    4.6*10.sup.7 c.f.u. of strain 3290/91(A)

    4.4*10.sup.7 c.f.u. of strain 3290/91(K)

At 32 days of age, 9 vaccinated and 9 non-vaccinated animals werechallenged into the right thoracic airsac with 0.2 ml of thechallenge-mixture, mentioned above as preparation 1. At 41 days of age,the animals were weighed and a post mortem was performed

At 35 days of age, 9 vaccinated and 8 non-vaccinated animals werechallenged into the right thoracic airsac with 0.2 ml of the challenge-mixture, mentioned above as preparation 2. At 41 days of age, theanimals were weighed and a post mortem was done.

Results A) Virulence of strain 3263/91 in chickens

The table 5 given below shows the virulence of strain 3263/91 depositedunder CBS 400.92 in broilers, determined by growth-retardation, when itis used as a live challenge-strain. Growth retardation is a result ofthe disease, and as such is a good indicator for the virulence ofpathogenic strains, and also for the efficacy of vaccination. The strainwas grown on egg-yolk as described under EXAMPLE II: preparation ofchallenge strains. Challenge material was brought directly into theairsacs, in a concentration of 1.2*10⁸ C.F.U. per animal.

                  TABLE 5                                                         ______________________________________                                        Comparison of growth-development in chickens infected                         with live strain 3262/91 and control group,                                   Challenge       strain 3263/91                                                                            control group                                     ______________________________________                                        Average weight  1100 (± 98)                                                                            1143 (± 110)                                   day 0                                                                         Average weight  1179 (± 132)                                                                           1478 (± 92)                                    day 8                                                                         Average weight  1684 (± 162)                                                                           1935 (± 91)                                    day 14                                                                        Average weight diff.                                                                           93 (± 114).sup.a                                                                       314 (± 64)                                    day 0-8                                                                       Average weight diff.                                                                           600 (± 165).sup.b                                                                      796 (± 74)                                    day 0-14                                                                      ______________________________________                                         .sup.a significantly different from the control group, p <0.005               .sup.b significantly different from the control group, p <0.05           

B) Virulence of strain 3263/91 and GGD 1261 in turkeys

The table 6 given below shows the virulence of strain 3263/91 depositedunder CBS 400.92 and the turkey strain GGD 1261 in turkeys, determinedby growth-retardation, when they are used as live challenge-strains. Thestrains were grown on egg-yolk as described under EXAMPLE II:preparation of challenge strains. Challenge material was broughtdirectly into the airsacs, in a concentration of 5*10⁸ C.F.U. per animalat an age of 32 days. Eleven days after the infection, the turkeys weresacrificed.

                  TABLE 6                                                         ______________________________________                                        Comparison of growth-development in turkeys infected                          with live strain 3262/91, live strain GGD 1261 and control                    group.                                                                        Challenge strain 3263/91                                                                            strain GGD control group                                ______________________________________                                        Average daily                                                                           65.sup.a    56.sup.b   82                                           Weight gain                                                                   after 11                                                                      days (grams)                                                                  ______________________________________                                         .sup.a significantly different from the control group, p <0.001               .sup.b significantly different from the control group, p <0.001          

c) Vaccination-challenge experiments in relation with pathology

1) The non-vaccinated group of 9 birds, challenged with the mixture ofB.H.I.-cultures showed swollen heads or swollen wattles in 5 out of 9animals, while the airsacs of 7 of the birds showed minor yellowishspots restricted to only the injection-site. The vaccinated group of 9birds, challenged with the mixture of B.H.I.-. cultures showed swollenheads or swollen wattles only in 2 out of 9 birds, while the airsac ofall the birds was fully clear, and showed no spots at the injectionsite.

2) The control group of 9 birds, challenged with the mixture of egg-yolkcultures showed minor yellowish spots at the injection site in 3 out of8 birds. In 3 birds some turbidity of the airsacs was seen, while onebird had a creamy exudate in all airsacs. From this exudate, a pureculture of bacteria fully homologous to the deposited strain could begrown. Only one bird showed no reaction at all. The vaccinated group ofnine birds, challenged with the mixture of egg-yolk cultures showedhealthy birds with very clear airsacs without spots at theinjection-site.

D) Vaccination-challenge experiments in relation with daily weight-gain

In table 7, the average daily weight gain of chickens over a period of34 days is given. It is easily seen from this table on the basis ofdifferences in daily weight gain, that turkey strain GGD 1261 ispathogenic for chickens. Most important however is the notice, thatvaccination with the deposited strain 3263/91 gives protection againstGGD-1261 challenge.

                  TABLE 7                                                         ______________________________________                                        vaccination challenge experiments in chickens                                 with vaccines based on strain 3263/91 and GGD 1261                            ______________________________________                                        GROUP               AVG       STD    n                                        ______________________________________                                        control             60        14     10                                       chall GGD-1261      42         9     10                                       vacc. GGD-1261 and hom. chall.                                                                    61        24     10                                       vacc 3263/91 + chall GGD-1261                                                                     60        12     10                                       ______________________________________                                        GROUPS            P =                                                         ______________________________________                                        Group 1 vs Group 2                                                                              <0.005                                                      Group 1 vs Group 3                                                                              >0.05                                                       Group 2 vs Group 3                                                                              <0.025                                                      Group 2 vs Group 4                                                                              <0.005                                                      ______________________________________                                         AVG = average, STD = standard deviation, n = number of animals.          

In conclusion, the results show, that strain 3263/91, deposited underCBS 400.92 is in its live form a virulent challenge-strain, capable ofinducing growth-retardation, swollen wattles, swollen heads andairsacculitis. When used in a vaccine-preparation, inactivated cells ofthe novel bacteria are capable of inducing protection against clinicalsymptoms, caused by live strains in both chickens and turkeys.

We claim:
 1. A vaccine effective against a disease of the lowerrespiratory tract in poultry, comprising inactivated Ornithobacteriumrhinotracheale.
 2. The vaccine according to claim 1, wherein the vaccinealso comprises an adjuvant.
 3. The vaccine according to claim 2, furthercomprising at least one other antigen from a virus or microorganismpathogenic to poultry.
 4. The vaccine according to claim 3, wherein themicroorganism or virus is selected from the group consisting ofInfectious Bronchitis Virus, Newcastle Disease Virus, Infectious BursalDisease, Chicken Anemia agent, Avian Reovirus, Mycoplasma gallisepticum,Turkey Rhinotracheitis Virus, Haemophilus paragallinarum, ChickenPoxvirus, Avian Encephalomyelitis Virus, Fowl Cholera and E. coli. 5.The vaccine according to claim 1, wherein it further comprises at leastone other antigen from a virus or microorganism pathogenic to poultry.6. A vaccine according to claim 5, wherein the microorganism or virus isselected from the group consisting of Infectious Bronchitis Virus,Newcastle Disease Virus, Infectious Bursal Disease, Chicken Anemiaagent, Avian Reovirus, Mycoplasma gallisepticum, Turkey RhinotracheitisVirus, Haemophilus paragallinarum, Chicken Poxvirus, AvianEncephalomyelitis Virus, Fowl Cholera and E. coli.
 7. The vaccineaccording to claim 1, wherein the Ornithobacterium rhinotracheale isthat which is deposited at the Centraalbureau vor Schimmecultures (CBS)under Deposit No. 400.92.
 8. The vaccine according to claim 7, whereinthe vaccine also comprises an adjuvant.
 9. The vaccine according toclaim 8, further comprising at least one other antigen from a virus ormicroorganism pathogenic to poultry.
 10. The vaccine according to claim9, wherein the microorganism or virus is selected from the groupconsisting of Infectious Bronchitis Virus, Newcastle Disease Virus,Infectious Bursal Disease, Chicken Anemia agent, Arian Reovirus,Mycoplasma gallisepticum, Turkey Rhinotracheitis virus, Haemophilusparagallinarum, Chicken Poxvirus, Avian Encephalomyelitis Virus, FowlCholera and E. coli.
 11. The vaccine according to claim 7, which furthercomprises at least one other antigen from a virus or microorganismpathogenic to poultry.
 12. A vaccine according to claim 11, wherein themicroorganism or virus is selected from the group consisting ofInfectious Bronchitis virus, Newcastle Disease Virus, Infectious BursalDisease, Chicken Anemia agent, Arian Reovirus, Mycoplasma gallisepticum,Turkey Rhinotracheitis Virus, Haemophilus paragallinarum, ChickenPoxvirus, Avian Encephalomyelitis Virus, Fowl Cholera and E. coli.
 13. Amethod of immunizing poultry against bacterial respiratory disease,comprising administering an immunologically effective amount of avaccine according to claim
 1. 14. A method of immunizing poultry againstbacterial respiratory disease, comprising administering animmunologically effective amount of a vaccine according to claim 7.